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Agricultural Sci. J. 41(3/1)(Suppl.): 557-560 (2010)
ว. วิทย์. กษ. 41(3/1)(พิเศษ): 557-560 (2553)
สารออกฤทธิ์ทางชีวภาพจากราเอนโดไฟท์ ของพืชในวงศ์ Stemonaceae ในการควบคุมแบคทีเรี ย ทาให้ เกิดโรคในพืช : สูตรอาหารที่เหมาะสม Bioactive Compounds from Endophytic Fungi of Stemona spp. for Plant Pathogenic Bacteria Control : Medium Optimization ชัยวัฒน์ บุญมากาศ1 ทรงศิลป์ พจน์ ชนะชัย1 ศันสนลักษณ์ รั ชฎาวงศ์ 1 และ ทวีรัตน์ วิจติ รสุนทรกุล1 Boonmakard, C.1, Photchanachai, S.2, Rachadawong, S.1 and Vichitsoonthonkul, T.1
Abstract Fusarium sp. L LSB49-1, an endophytic fungus, isolated from S. burkillii leave, produced antibacterial activity in M1D medium against Erwinia carotovora, Pseudomonas solanacearum biovar.2, Pseudomonas solanacearum biovar.4, and Xanthomonas citri. Production of bioactive compounds from Fusarium sp. L LSB49-1 was optimized in different substrates including carbon sources and nitrogen sources. Fructose, glucose, sucrose, molasses, and cassava starch were used as carbon sources. Ammonium tartrate, ammonium chloride, ammonium sulphate and urea were used as nitrogen sources in M1D medium. Sucrose and ammonium tartrate could enhance antibacterial compound production in Fusarium sp. LLSB49-1 at 28°C,150 rpm. Keywords: antiphyotogenic bacteria compounds, fungal endophyte, Stemona spp, medium optimization บทคัดย่ อ Fusarium sp. LLSB49-1 ที่แยกได้ จากใบ ของ Stemona burkillii สามารถสร้ างสารยับยั ้งเชื ้อแบคทีเรียก่อโรคในพืช เศรษฐกิจของไทย 3 ชนิดได้ แก่ Erwinia carotovora, Pseudomonas solanacearum, และ Xanthomonas citri วัตถุประสงค์ ของงานนี ้คือการหาสารอาหารที่เหมาะสมที่สดุ ในการสร้ างสารออกฤทธิ์ทางชีวภาพ ของ Fusarium sp. LLSB49-1 ได้ แก่ แหล่งคาร์ บอนและไนโตรเจน พบว่าเมื่อใช้ ซูโครส, ฟรุคโตส, กลูโคส, กากน ้าตาล และแป้งมันสาปะหลังเปน นแหล่งคาร์ บอน และ ใช้ แอมโมเนียมทาร์ เทรท, แอมโมเนียมคลอไรด์ , แอมโมเนียมซัลเฟต และยูเรี ยเปน นแหล่งไนโตรเจนในอาหาร พบว่า Fusarium sp. LLSB49-1 สามารถสร้ างสารที่ยบั ยั ้งแบคทีเรียได้ ดีที่สดุ เมื่อเลี ้ยงในอาหารที่มีน ้าตาลซูโครสและแอมโมเนียมทาร์ เทรทเปน น แหล่งคาร์ บอนและไนโตรเจนตามลาดับ เมื่อเลี ้ยงเชื ้อที่อณ ุ หภูมิ 28 องศาเซลเซียส เขย่าที่ความเรนว 150 รอบต่อนาที คาสาคัญ: สารต้ านแบคทีเรี ยทาให้ เกิดโรคในพืช ราเอนโดไฟท์ พืชในวงศ์ Stemonaceae สูตรอาหารเลี ้ยงเชื ้อที่เหมาะสม Introduction Fusarium sp. L LSB49-1, an endophytic fungus isolated from S. burkillii leave, was reported as a high capacity producer of active metabolites against some bacterial plant pathogens (i.e. E. caratovora, X. citri, and P. solanacearum) (Boonmakard, 2009). Antimicrobial compounds are secondary metabolites and their production is regulated by culture condition and medium components such as carbon sources, nitrogen sources, phosphate, and trace elements (Betina, 1994). Carbon and nitrogen sources which are favorable for growth may not be beneficial to secondary metabolite production (Demain, 1992). The aim of this work is to optimize carbon sources and nitrogen sources for antibacterial compound production by Fusaium sp. L LSB49-1.
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สายวิชาเทคโนโลยีชีวภาพ คณะทรัพยากรชีวภาพและเทคโนโลยี มหาวิทยาลัยเทคโนโลยีพระจอมเกล้ าธนบุรี กรุงเทพฯ 10150 Division of Biotechnology, School of Bioresources and Technology, King Mongkut’s University of Technology, Thonburi, Bangkok 10150 2 สายวิชาเทคโนโลยีหลังการเก็บเกี่ยว คณะทรัพยากรชีวภาพและเทคโนโลยี มหาวิทยาลัยเทคโนโลยีพระจอมเกล้ าธนบุรี กรุงเทพฯ 10150 2 Division of Post Harvest Technology, School of Bioresources and Technology, King Mongkut’s University of Technology, Thonburi, Bangkok 10150 1
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ว. วิทยาศาสตร์ เกษตร
Materials and Methods Carbon source in 100 ml of M1D medium (Stroble et al., 2002) was replaced individually by evaluated C sources, namely, glucose, fructose, molasses, sucrose, and starch at a concentration of 30 g/L. Four different sources of nitrogens were used, i.e. ammonium chloride (NH4Cl), ammonium sulfate ((NH4)2SO4), ammonium tartrate and urea. The nitrogen content of each source was equivalent to the amount of nitrogen in original ammonium tartrate concentration in M1D medium. Spore suspension of Fusarium sp. L LSB49-1 was inoculated at a final concentration of 106 spores/ml of medium. The cultures were incubated at 28oC, 150 rpm. Biomass, sugar residual, pH of culture broth and antibacterial activity were determined as described in Boonmakard (2009). Results and Discussion There were significant differences in growth, sugar consumption and pH change in the culture of Fusarium sp. L LSB49-1 when various C sources were supplemented separately in growth medium as shown in Fig 1. Cassava starch produced least biomass (1.7 g/L) and antibacterial agent was not detected in this medium. This could be due to low efficient enzyme(s) in starch metabolism and/or high pH of the medium. Fructose, glucose and molasses were more rapidly metabolized and less antibacterial agent production by Fusarium sp. L LSB49-1 when compared to sucrose medium. This negative effect on secondary metabolism could be the result of carbon catabolite repression (CRC) of rapidly utilizable C sources which has been found in fungal secondary metabolism such as in penicillin biosynthesis by Penicillium chrysosporium (Ravilla et al.,1984). Sucrose was a more beneficial C source since it had to be converted to glucose and fructose before assimilation. As a result, it was metabolized more slowly than that of other sugars used in this study. Four tested nitrogen sources were all favorable to fungal growth (Figure 3) but they exhibited different effects on antibacterial compound production (Figure 4). Highest antibacterial activities was obtained in ammonium tartrate medium (Figure 4). The results also revealed that rapid assimilation of sucrose in the media containing (NH4)2SO4 and NH4Cl resulting in high growth rates and inhibitory effect on secondary metabolite formation (data not shown). It has been known that culture condition providing high specific growth rate usually suppresses secondary metabolism in fungi (Demain, 1986). However, urea also favoured high growth rate but it exerted lower level of interference to antibacterial agent production in Fusarium sp. L LSB49-1 because the antibacterial activities were detectable with delayed time course when compared to those found in ammonium tartrate medium (Figure 4). It should be noted that the pH value in urea medium was as high as 9.1 at day 0 as a result of urea hydrolysis during sterilization process and dropped to about 6.1 at day 4 and then gradually rose to 8.0 at the end of experiment. In contrast, pH of (NH4)2SO4 and NH4Cl media markedly decreased with the consumption sucrose (Figure 4) from the initial pH value of 4.3 and 4.7 to pH 2.3 and 2.2, respectively, at day 2. External pH has been found to affect expression of many secondary metabolism genes in fungi. pcbC and ipnA , structural genes in penicillin biosynthesis in P. chrysosporium and A. nidulans , respectively, are suppressed by low pH (Espeso et al., 1993) where as structural genes in aflatoxin and sterigmatocystin biosynthesis in Aspergillus spp.are induced by acidic pH (Keller et al., 1997). These results suggested that the environment factor like pH, were found to have profound influence on antimicrobial compound production in Fusarium sp. L LSB49-1. Summary Bioactive compound production in Fusarium sp. LLSB49-1 is subjected to C and N sources in the medium. Sucrose and ammonium tartrate could enhance antibacterial compound production in this fungus. The pH of culture broth seems to play impotant role in secondary metabolism in this endophytic fungi.
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Figure 1 Effects of various carbon sources i.e. (a) glucose, (b) fructose, (c) molasses, (d) cassava starch and (e) sucrose on the growth, pH and residual sugar of culture of Fusarium sp. L LSB49-1. P. solanacearum biovar.2
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Figure 4 Effects of various nitrogen sources on growth and bioactive compound production of Fusarium sp. L LSB49-1 in different antibacterial activity against bacterial plant pathogen (·····, cell dry weight; — , inhibition zone). Literature cited Betina, V., 1994, Physiological Regulation of Secondary Metabolism, In: Betina V., editor. Bioactive secondary metabolite of microorganisms: process in industrial microbiology. Vol 30. Amsterdam & New York , Elsevier Science, p. 66-80. Boonmakard, C., 2009, Screening for Bioactive Compounds from Endophytic Fungi of Stemona spp. for Plant Pathogenic Bacteria Control, M. Sc.Thesis, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi. Demain, A.L., Microbial Secondary Metabolism: a New Theoretical Frontier for Academia, a New Opportunity for Industry, In : Chadwick, D.J., Whealan, D., editors. Secondary Metabolites: Their Function and Evolution. Chichester, UK: John Wiley & Sons; 1992. p. 9. Espeso, E.A., Tilburn, J., Arst, H.N. and Penalva., M.A., 1993, pH Regulation is a Major Determinant in Expression of a Fungal Penicillin Biosynthesis Gene, EMBO Journal, 12: 3947-3956. Keller, N.P., Nesbit, C., Sarr, B., Phillips, T.D. and Burrow, G.B., 1997, pH Regulation of Sterigmatocystin and Aflatoxin Biosynthesis in Aspergillus spp., Phytopathology, 87: 643-648. Revilla, G., Villanueva, J.R., Leungo, J.M. and Martin, J.F., 1984, Carbon Catabolite Repression of Penicillin Biosynthesis by Penicillium chrysosporium, Journal of Antibiotics, 37: 781-789.
ว. วิทย์. กษ. 41(3/1)(พิเศษ): 557-560 (2553)
สารออกฤทธิ์ทางชีวภาพจากราเอนโดไฟท์ ของพืชในวงศ์ Stemonaceae ในการควบคุมแบคทีเรี ย ทาให้ เกิดโรคในพืช : สูตรอาหารที่เหมาะสม Bioactive Compounds from Endophytic Fungi of Stemona spp. for Plant Pathogenic Bacteria Control : Medium Optimization ชัยวัฒน์ บุญมากาศ1 ทรงศิลป์ พจน์ ชนะชัย1 ศันสนลักษณ์ รั ชฎาวงศ์ 1 และ ทวีรัตน์ วิจติ รสุนทรกุล1 Boonmakard, C.1, Photchanachai, S.2, Rachadawong, S.1 and Vichitsoonthonkul, T.1
Abstract Fusarium sp. L LSB49-1, an endophytic fungus, isolated from S. burkillii leave, produced antibacterial activity in M1D medium against Erwinia carotovora, Pseudomonas solanacearum biovar.2, Pseudomonas solanacearum biovar.4, and Xanthomonas citri. Production of bioactive compounds from Fusarium sp. L LSB49-1 was optimized in different substrates including carbon sources and nitrogen sources. Fructose, glucose, sucrose, molasses, and cassava starch were used as carbon sources. Ammonium tartrate, ammonium chloride, ammonium sulphate and urea were used as nitrogen sources in M1D medium. Sucrose and ammonium tartrate could enhance antibacterial compound production in Fusarium sp. LLSB49-1 at 28°C,150 rpm. Keywords: antiphyotogenic bacteria compounds, fungal endophyte, Stemona spp, medium optimization บทคัดย่ อ Fusarium sp. LLSB49-1 ที่แยกได้ จากใบ ของ Stemona burkillii สามารถสร้ างสารยับยั ้งเชื ้อแบคทีเรียก่อโรคในพืช เศรษฐกิจของไทย 3 ชนิดได้ แก่ Erwinia carotovora, Pseudomonas solanacearum, และ Xanthomonas citri วัตถุประสงค์ ของงานนี ้คือการหาสารอาหารที่เหมาะสมที่สดุ ในการสร้ างสารออกฤทธิ์ทางชีวภาพ ของ Fusarium sp. LLSB49-1 ได้ แก่ แหล่งคาร์ บอนและไนโตรเจน พบว่าเมื่อใช้ ซูโครส, ฟรุคโตส, กลูโคส, กากน ้าตาล และแป้งมันสาปะหลังเปน นแหล่งคาร์ บอน และ ใช้ แอมโมเนียมทาร์ เทรท, แอมโมเนียมคลอไรด์ , แอมโมเนียมซัลเฟต และยูเรี ยเปน นแหล่งไนโตรเจนในอาหาร พบว่า Fusarium sp. LLSB49-1 สามารถสร้ างสารที่ยบั ยั ้งแบคทีเรียได้ ดีที่สดุ เมื่อเลี ้ยงในอาหารที่มีน ้าตาลซูโครสและแอมโมเนียมทาร์ เทรทเปน น แหล่งคาร์ บอนและไนโตรเจนตามลาดับ เมื่อเลี ้ยงเชื ้อที่อณ ุ หภูมิ 28 องศาเซลเซียส เขย่าที่ความเรนว 150 รอบต่อนาที คาสาคัญ: สารต้ านแบคทีเรี ยทาให้ เกิดโรคในพืช ราเอนโดไฟท์ พืชในวงศ์ Stemonaceae สูตรอาหารเลี ้ยงเชื ้อที่เหมาะสม Introduction Fusarium sp. L LSB49-1, an endophytic fungus isolated from S. burkillii leave, was reported as a high capacity producer of active metabolites against some bacterial plant pathogens (i.e. E. caratovora, X. citri, and P. solanacearum) (Boonmakard, 2009). Antimicrobial compounds are secondary metabolites and their production is regulated by culture condition and medium components such as carbon sources, nitrogen sources, phosphate, and trace elements (Betina, 1994). Carbon and nitrogen sources which are favorable for growth may not be beneficial to secondary metabolite production (Demain, 1992). The aim of this work is to optimize carbon sources and nitrogen sources for antibacterial compound production by Fusaium sp. L LSB49-1.
1
สายวิชาเทคโนโลยีชีวภาพ คณะทรัพยากรชีวภาพและเทคโนโลยี มหาวิทยาลัยเทคโนโลยีพระจอมเกล้ าธนบุรี กรุงเทพฯ 10150 Division of Biotechnology, School of Bioresources and Technology, King Mongkut’s University of Technology, Thonburi, Bangkok 10150 2 สายวิชาเทคโนโลยีหลังการเก็บเกี่ยว คณะทรัพยากรชีวภาพและเทคโนโลยี มหาวิทยาลัยเทคโนโลยีพระจอมเกล้ าธนบุรี กรุงเทพฯ 10150 2 Division of Post Harvest Technology, School of Bioresources and Technology, King Mongkut’s University of Technology, Thonburi, Bangkok 10150 1
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ปี ที่ 41 ฉบับที่ 3/1 (พิเศษ) กันยายน-ธันวาคม 2553
ว. วิทยาศาสตร์ เกษตร
Materials and Methods Carbon source in 100 ml of M1D medium (Stroble et al., 2002) was replaced individually by evaluated C sources, namely, glucose, fructose, molasses, sucrose, and starch at a concentration of 30 g/L. Four different sources of nitrogens were used, i.e. ammonium chloride (NH4Cl), ammonium sulfate ((NH4)2SO4), ammonium tartrate and urea. The nitrogen content of each source was equivalent to the amount of nitrogen in original ammonium tartrate concentration in M1D medium. Spore suspension of Fusarium sp. L LSB49-1 was inoculated at a final concentration of 106 spores/ml of medium. The cultures were incubated at 28oC, 150 rpm. Biomass, sugar residual, pH of culture broth and antibacterial activity were determined as described in Boonmakard (2009). Results and Discussion There were significant differences in growth, sugar consumption and pH change in the culture of Fusarium sp. L LSB49-1 when various C sources were supplemented separately in growth medium as shown in Fig 1. Cassava starch produced least biomass (1.7 g/L) and antibacterial agent was not detected in this medium. This could be due to low efficient enzyme(s) in starch metabolism and/or high pH of the medium. Fructose, glucose and molasses were more rapidly metabolized and less antibacterial agent production by Fusarium sp. L LSB49-1 when compared to sucrose medium. This negative effect on secondary metabolism could be the result of carbon catabolite repression (CRC) of rapidly utilizable C sources which has been found in fungal secondary metabolism such as in penicillin biosynthesis by Penicillium chrysosporium (Ravilla et al.,1984). Sucrose was a more beneficial C source since it had to be converted to glucose and fructose before assimilation. As a result, it was metabolized more slowly than that of other sugars used in this study. Four tested nitrogen sources were all favorable to fungal growth (Figure 3) but they exhibited different effects on antibacterial compound production (Figure 4). Highest antibacterial activities was obtained in ammonium tartrate medium (Figure 4). The results also revealed that rapid assimilation of sucrose in the media containing (NH4)2SO4 and NH4Cl resulting in high growth rates and inhibitory effect on secondary metabolite formation (data not shown). It has been known that culture condition providing high specific growth rate usually suppresses secondary metabolism in fungi (Demain, 1986). However, urea also favoured high growth rate but it exerted lower level of interference to antibacterial agent production in Fusarium sp. L LSB49-1 because the antibacterial activities were detectable with delayed time course when compared to those found in ammonium tartrate medium (Figure 4). It should be noted that the pH value in urea medium was as high as 9.1 at day 0 as a result of urea hydrolysis during sterilization process and dropped to about 6.1 at day 4 and then gradually rose to 8.0 at the end of experiment. In contrast, pH of (NH4)2SO4 and NH4Cl media markedly decreased with the consumption sucrose (Figure 4) from the initial pH value of 4.3 and 4.7 to pH 2.3 and 2.2, respectively, at day 2. External pH has been found to affect expression of many secondary metabolism genes in fungi. pcbC and ipnA , structural genes in penicillin biosynthesis in P. chrysosporium and A. nidulans , respectively, are suppressed by low pH (Espeso et al., 1993) where as structural genes in aflatoxin and sterigmatocystin biosynthesis in Aspergillus spp.are induced by acidic pH (Keller et al., 1997). These results suggested that the environment factor like pH, were found to have profound influence on antimicrobial compound production in Fusarium sp. L LSB49-1. Summary Bioactive compound production in Fusarium sp. LLSB49-1 is subjected to C and N sources in the medium. Sucrose and ammonium tartrate could enhance antibacterial compound production in this fungus. The pH of culture broth seems to play impotant role in secondary metabolism in this endophytic fungi.
ว. วิทยาศาสตร์ เกษตร
ปี ที่ 41 ฉบับที่ 3/1 (พิเศษ) กันยายน-ธันวาคม 2553
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Figure 1 Effects of various carbon sources i.e. (a) glucose, (b) fructose, (c) molasses, (d) cassava starch and (e) sucrose on the growth, pH and residual sugar of culture of Fusarium sp. L LSB49-1. P. solanacearum biovar.2
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Figure 2 Effects of various carbon sources on growth and bioactive compound production of Fusarium sp. L LSB49-1 in different antibacterial activity against bacterial plant pathogen (·····, cell dry weight; — , inhibition zone).
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ปี ที่ 41 ฉบับที่ 3/1 (พิเศษ) กันยายน-ธันวาคม 2553 P. solanacearum biovar.4
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ว. วิทยาศาสตร์ เกษตร
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Figure 4 Effects of various nitrogen sources on growth and bioactive compound production of Fusarium sp. L LSB49-1 in different antibacterial activity against bacterial plant pathogen (·····, cell dry weight; — , inhibition zone). Literature cited Betina, V., 1994, Physiological Regulation of Secondary Metabolism, In: Betina V., editor. Bioactive secondary metabolite of microorganisms: process in industrial microbiology. Vol 30. Amsterdam & New York , Elsevier Science, p. 66-80. Boonmakard, C., 2009, Screening for Bioactive Compounds from Endophytic Fungi of Stemona spp. for Plant Pathogenic Bacteria Control, M. Sc.Thesis, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi. Demain, A.L., Microbial Secondary Metabolism: a New Theoretical Frontier for Academia, a New Opportunity for Industry, In : Chadwick, D.J., Whealan, D., editors. Secondary Metabolites: Their Function and Evolution. Chichester, UK: John Wiley & Sons; 1992. p. 9. Espeso, E.A., Tilburn, J., Arst, H.N. and Penalva., M.A., 1993, pH Regulation is a Major Determinant in Expression of a Fungal Penicillin Biosynthesis Gene, EMBO Journal, 12: 3947-3956. Keller, N.P., Nesbit, C., Sarr, B., Phillips, T.D. and Burrow, G.B., 1997, pH Regulation of Sterigmatocystin and Aflatoxin Biosynthesis in Aspergillus spp., Phytopathology, 87: 643-648. Revilla, G., Villanueva, J.R., Leungo, J.M. and Martin, J.F., 1984, Carbon Catabolite Repression of Penicillin Biosynthesis by Penicillium chrysosporium, Journal of Antibiotics, 37: 781-789.
